1. Field of the Invention
The present invention relates to a chamber and a device for effectively performing cell fusion and a method for cell fusion using the same.
2. Description of the Related Art
In the prior art, a chemical fusion method mainly using polyethyleneglycol (PEG) is used as a cell fusion technique for fusing different cells to obtain a hybridoma. However, this method has the following problems to be solved, for example: (i) PEG exhibits a strong toxicity against cells; (ii) a great deal of effort is required in finding the optimal conditions for cell fusion with respect to the polymerization degree or amount of PEG added, or the like; (iii) an advanced technique is required for carrying out cell fusion, and so only a person skilled in a particular technique can use this method; (iv) since contact between two cells accidentally occurs, it is difficult to control cell fusion of two cells, and so the probability of successful cell fusion is extremely low.
In contrast, an electrical cell fusion method has an advantage in that an advanced technique is not required, cell fusion can be easily and effectively carried out, toxicity is almost not exhibited against cells, and cells can be fused with high activity. The electrical cell fusion method was established by Zimmermann in 1981 in Western Germany and the theory thereof is as follows. An alternating voltage is applied to parallel electrodes, and cells are introduced therein, as a result of which the cells are drawn toward the area having higher current density and thus linearly linked together. The state in which cells are linearly linked together is generally referred to as a pearl chain. In this state, a voltage pulsed direct current is applied between electrodes at intervals of a few micro seconds to several tens of micro seconds, as a result of which electrical conductance of the cellular membrane instantly decreases, and thereby cellular membranes constituted by a lipid bilayer are reversibly disrupted and then reconstituted, and thus cell fusion is performed.
As the electrical fusion method, a microelectrode method and a parallel electrode method are mainly used. The microelectrode method is a method in which two cells are fused by picking up the cells by operating a micromanipulator while observing them using a microscope, and then applying a voltage pulsed direct current thereto. This method enables extremely reliable cell fusion. Moreover, an electrode of the micromanipulator has been reported (see, for example, Patent Document 1 (Japanese Examined Patent Application, Second Publication No. H 7-40914)). However, the microelectrode method requires lengthy procedure, the operation of the micromanipulator requires skill, and the method is not practical in view of dealing with a large number of cells. In contrast, the parallel electrode method is a method in which a voltage pulsed direct current is applied to a pearl chain formed by plural cells subjected to dielectrophoresis, and the handling thereof is easy. However, since the plural cells linearly linked together are fused, contact between two cells accidentally occurs, and so there is a problem in that it is difficult to reliably control cell fusion of two cells.
In order to solve the problem of the parallel electrode method, a cell fusion chamber composed of a pair of electrodes formed by a conductor and placed opposite to each other in a cell fusion region and an insulator placed between the pair of electrodes and having a fine pore passing therethrough in the direction of the pair of electrodes has been reported (see, for example, Patent Document 2 (Japanese Examined Patent Application, Second Publication No. H 7-4218)).
FIG. 1 is a schematic diagram showing a cross-sectional view of the above-mentioned cell fusion chamber. In FIG. 1, electrodes (2) formed of a conductor are placed at both sides of a cell fusion region (1) in a cell fusion chamber formed of a resin, for example. To these electrodes, an electronic power supply (4) placed outside is connected through a conductor (3). The electronic power supply (4) is composed of an alternating-current power supply (5) outputting a high-frequency alternating voltage with an electrical field strength of approximately 400 V/cm to 700 V/cm and a frequency of approximately 1 MHz, a direct current pulsed power supply (6) outputting a voltage pulsed direct current with an electrical field strength of approximately 7 kV/cm and a pulse width of 50μ seconds, and a switch (7) having a switching device which changes electrical connection with the electrodes between the alternating-current power supply (5) and the direct current pulsed power supply (6).
As the waveform of the alternating voltage output from the alternating-current power supply (5), a sine-wave waveform is generally used if not otherwise specified. The cell fusion chamber is divided into two compartments by a partition wall (35) formed by an insulator such as a silicone resin or the like. In the partition wall (35), a fine pore (9) having a minimum diameter of 1 μm to several tens of μm is formed. Cell A (10) and Cell B (11) are each contained in a cell suspension placed in the cell fusion region of the cell fusion chamber.
The performance in the above-mentioned example will be explained using FIG. 2 to FIG. 4. First of all, the switch (7) of the electronic power supply (4) is connected to the alternating-current power supply (5), which outputs a high-frequency voltage with an electrical field strength of approximately 400 V/cm to 700 V/cm and a frequency of 1 MHz. In this state, lines of electric force (12) concentrate at the fine pore (9), as shown in FIG. 2. Cell A (10) and Cell B (11) are affected by a dielectrophoretic force caused by the lines of electric force (12), and they are transferred to near the center portion of the fine pore (9), as shown in FIG. 3. Thus, Cell A (10) and Cell B (11) come into contact with each other. Next, the switch (7) of the electronic power supply (4) is switched to the direct current pulsed power supply (6). In Cell A (10) and Cell B (11), which are left in the state as shown in FIG. 3, the cellular membranes thereof are reversibly disrupted by the voltage pulsed direct current at the contact point of Cell A (10) and Cell B (11), and then reconstituted. Thus, a fused cell (hybridoma) is produced, as shown in FIG. 4. According to this, Cell A (10) and Cell B (11) can be fused at the fine pore.
However, the method for cell fusion using the cell fusion chamber disclosed in Patent Document 2 has a problem in that when the diameter of the fine pore is larger than those of Cell A (10) and Cell B (11), the probability of Cell A (10) and Cell B (11) making contact at the fine pore (9) in the same direction as those of the lines of electric force as shown in FIG. 30 becomes low, and so the probability of cell fusion decreases. On the other hand, when the diameter of the fine pore is smaller than those of Cell A (10) and Cell B (11), although both of the cells are trapped by the fine pore (9), make contact with each other, and are fused, the fused cell cannot be separated from the fine pore (9) as shown in FIG. 31. Accordingly, when the fused cell is collected by pulling it by force, the fused cell may be destroyed.
Moreover, the method for cell fusion using the cell fusion chamber disclosed in Patent Document 2 has another problem in that it is difficult to fix the two cells in the fine pore at the same time. For example, when a cell suspension containing Cell B (11) is introduced in the cell fusion chamber so as to place Cell B (11) in the fine pore after Cell A (10) is placed in the fine pore, Cell A (10) placed in the fine pore in advance is separated from the fine pore by introducing the cell suspension. Also, fixation of Cell A (10) and Cell B (11) in the fine pore at the same time requires skill and is quite difficult. Moreover, when plural cells are fused at the same time according to the method disclosed in Patent Document 2, plural pairs of two cells are required to be fixed in plural fine pores formed in an array state on an insulator.
The array means the state in which plural fine pores are disposed at even longitudinal and horizontal intervals. However, there is a problem in that when an alternating-current power supply is connected so as to fix cells in the fine pores, some of the fine pores include a concentration of plural cells fixed therein, while some of the fine pores includes no cells fixed therein. Accordingly, it is quite difficult to fuse the objective plural pairs of two cells in the plural fine pores formed in the array state.
In contrast, a method in which cells are singly fixed in plural fine pores formed in an array state has been reported (see, for example, Patent Document 3 (Japanese Patent Granted Publication No. 3723882)). According to the method disclosed in Patent Document 3, a step of adding a suspension containing plural cells to cover plural fine pores (corresponding to “microwells” in Patent Document 3) of which the inside diameter and depth are within a range from equal to twice as large as those of the cells (corresponding to “test lymphocytes”) and waiting until the cells sink in the fine pores, and a step of washing out cells outside the fine pores are repeatedly conducted so as to fix a single cell in each fine pore.
However, the method in which a single cell is fixed in each fine pore as disclosed in Patent Document 3 has problems in that a long waiting time, approximately 5 minutes, is required until the cell sinks by gravity, the manipulation thereof is complicated and requires additional times for repeatedly conducting the steps of waiting until the cells sink in the fine pores and washing out the cells outside the fine pores, and it is difficult to effectively use all cells because some of the cells may be lost during the step of washing out the cells outside the fine pores. In general, when cell fusion is carried out, it is preferable to shorten the treatment time as much as possible, for the purpose of maintaining the activity of the cells, and to prevent the loss of cells as much as possible, for the purpose of finding the specificity possessed by the respective cells.